Pigments, size, and distribution of Synechococcus in the North Atlantic and Pacific Oceans

Transcription

1 Limnol. Oceanogr., 35(l), 1990,45-S , by the American Society of Limnology and Oceanography, Inc. Pigments, size, and distribution of Synechococcus in the North Atlantic and Pacific Oceans Robert J. Olson Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts Sallie W. Chisholm Ralph M. Parsons Laboratory, Massachusetts Institute of Technology, Cambridge Erik R. Zettler Woods Hole Oceanographic Institution E. Virginia Armbrust Massachusetts Institute of Technology Abstract Dual-beam flow cytometry was used to analyze the distribution and optical characteristics of Synechococcus in the North Atlantic and Pacific Oceans. The depth range over which Synechococcus cells were abundant was related to the depth of the nitrite maximum and the chlorophyll maximum, but was not significantly correlated with the depth of the surface isothermal layer. Dual-beam analysis of chromophore pigment types revealed that the majority of the populations were of the high-urobilin type; low-urobilin types, similar to the isolate WH7803, were found only in coastal waters where they almost always co-occurred with high-urobilin strains. Phycoerythrin fluorescence intensity per cell increased dramatically with depth in the lower euphotic zone at all stations; at some open-ocean stations, very deep cells were as much as 100 times brighter than those at the surface. The maximal fluorescence intensity per cell was about the same at the coastal and oceanic stations, and the depth of maximal fluorescence was closely related to the depth of the nitrite maximum. At most stations, fluorescence per cell was constant throughout the mixed layer, but at some open-ocean stations it decreased continuously to the surface. The latter pattern suggests that mixing rates in these areas are slow relative to the abilities of the cells to photoacclimate. A distinct diel pattern in forward-angle light scatter was observed in cells in the mixed layer over vast regions, which we hypothesize to be coupled to growth of the cells during daylight hours. Photosynthetic picoplankton, both procaryotic and eucaryotic, have been recognized in the last few years as important components of the phytoplankton in most of the world s oceans. Numerous studies have Acknowledgments We thank John Waterbury, Frederica Valois, and Diana Franks for providing cultures of Synechococcus, and for discussions and advice; Sheila Frankel, Alan Lewitus, Mark Gerath, and Heidi Sosik for technical support; Trevor Platt, Holger Jannasch, Craig Smith, Diane Stoecker, and Stanley Watson for providing ship time; and the captains and crews of the RVs Knorr, Gyre, Atlantis II, Oceanus, and CNS Hudson. This work was supported in part by NSF grants OCE , OCE , OCE , OCE , and CEE 82-l 1525 (to S.W.C.), OCE and OCE (to R.J.O.), and ONR contracts N K-066 1, 84-C-0278, and 87-K-0007 (to R.J.O. and S.W.C.). Woods Hole Oceanographic Institution contribution shown that cells passing a 3-pm filter constitute a significant proportion of the total biomass and production in many oceanic regimes (Saijo 1964; Berman 1975; Takahashi and Bienfang 1983). The phycoerythrin-containing unicellular cyanobacteria Synechococcus were the first picoplankton to be studied in detail (Waterbury et al. 1979; Johnson and Sieburth 1979) and the presence of this group has been documented extensively in warm oceans. These procaryotes are about 1,um in diameter and are easily recognized by epifluorescence microscopy because of the bright orange fluorescence of their major photosynthetic accessory pigment, phycoerythrin. Often they are more abundant numerically than the total eucaryotic phytoplankton, and estimates of the contribution of Synechococcus to primary production range from a few percent to more than half of the total for a water

2 46 Olson et al. column (Waterbury et al. 1986; Glover et al. 1986; Glover 1985; Iturriaga and Marra 1988). Small eucaryotes, including chlorophytes, prasinophytes, and eustigmatophytes, have recently been noted by several workers (Johnson and Sieburth 1982; Stockner and Antia 1986; Murphy and Haugen 1985), but their role has been less well studied, perhaps because they are very difficult to isolate and identify. Recently, even prochlorophyte picoplankton have been shown to be very abundant in the North Atlantic and Pacific Oceans (Chisholm et al. 1988; Li and Wood 1988). Flow cytometry has made it possible to study aspects of Synechococcus population dynamics difficult to approach by traditional methods (Wood et al. 1985; Olson et al. 1988; Li and Wood 1988). We have shown, for example, that the dominant pigment types in the open ocean are the high-phycourobilin types (Ong et al. 1984; Olson et al. 1988), many of which contain more urobilin than strains represented in culture collections. The absorptive properties of these high-plhycourobilin strains are not very different from those of the eucaryotic phytoplankton because they have a strong absorption peak at about 495 nm in addition to the 545-nm absorption peak of phycoerythrobilin. This pigment complement could explain why photosynthetic action spectra measured in oceanic waters do not yield the patterns one would expect from cells con- taining mainly phycoerythrobilin (Lewis et al. 1987). Here we expand our analyses of Synechococcus characteristics to broader areas of the oceans and examine in detail the lightscatter and fluorescence properties of populations as a function of depth and hydrographic regime. This analysis reveals patter:ns in the temporal and spatial structure in these populations that at present cannot be detected by other means. Materials and methods Sample collection and processing- Samples were analyzed with single-beam flow cytometry during cruises in the Caribbean and western North Atlantic (CNS Hudson, December 1984 and RV Knorr, December 1985). Dual-beam flow cytometry was used during cruises from Woods Hole, Massachusetts, to Dakar, Senegal (RV Gyre, September 1986), from Tampa, Florida, to San Diego, California, via the Panama Canal (RV Atlantis II, November-December 1986), from Woods Hole to the northern Sargasso Sea (RV Oceanus, June and September 1987), and to Georges Bank off Cape Cod, Massachusetts (July 1987) (Fig. 1). Water samples were taken with either PVC Niskin or Go-F10 bottl es for depth profiles (generally 12 depths between the surface and 200 m) or with plastic buckets when only surface samples were needed. Subsamples (3 ml) were prescreenecl through 53-pm Nitex mesh and 10 ~1 of a stock suspension of uniform fluorescent microspheres (0.9~pmdiam Fluoresbrite, Duke Scientific, and 2.1- pm-diam PR + NR, Pandex) were added to each sample before analysis by flow cytometry (Olson et al. 1988). chemical and physical parameter measurements - Samples for nitrate and nitrite analysis were either frozen or analyzed immediately according to Strickland and Par- sons (1972). Chlorophyll was determined fluorometrically after filtering water samples through either 0.4~pm-pore-diameter Nuclepore or GF/F glass-fiber filters and ex-,tracting overnight in cold 90% acetone (Strickland and Parsons 1972). Temperature profiles were obtained from an XBT or CTD. Since sampling was done at night on several of the cruises, and light measurements were not always possible at stations sampled during the day, we have used the depth of the nitrite maximum as an alternative measure of the euphotic zone depth (Voituriez and Herbland 1977; Herbland and Voituriez 1979). Flow cytometry measurements and data analysis-a Coulter EPICS V flow cytometer was used for all analyses, as described by Olson et al. (1988), except that on the Atlantis II and Oceanus cruises the laser spot size was reduced to 16 x 40 pm to increase sensitivity. We measured forward light scatter (FLS, an indicator of size), orange fluorescence from p hycoerythrin ( nm, PE), and red fluorescence from chlorophyll ( nm, Chl) after excitation by 488-nm light. On the last three cruises we used dual-beam flow cytometry

3 Synechococcus characteristics 47 2o O0 W llo 9o 7o 5o 3o lo0 Fig. 1. Flow cytometry of surface concentrations (cells ml-l) of Synechococcus. The size of the circles at the various locations indicates the relative abundance of Synechococcus. Filled symbols represent stations at which detailed profiles will be illustrated as examples of coastal and open-ocean situations (see Figs. 4, 9, II). Note that these designations were chosen partly on the basis of similarity of cell characteristics among the groups of stations rather than strictly geographically. to obtain a two-point fluorescence excitation spectrum (blue/green excitation ratio) for each cell, using both the 488- and 515- nm laser lines. This approach allowed us to determine the phycoerythrin chromophore composition of Synechococcus; i.e. we could distinguish those cells with relatively high phycourobilin content (PUB, absorbing at 495 nm) from those with little or no PUB, in which phycoerythrobilin (PEB, absorbing at 545 nm) is the dominant chromophore (Olson et al. 1988). When using single-beam excitation, we obtained three signals from each cell, while for dual-beam measurements we obtained five signals. As described in Olson et al. (1988), all the signals were stored in list mode and were analyzed by plotting two parameters at a time in a twodimensional histogram, after gating on a third parameter if desired. Numbers and geometric mean values for the light scatter and fluorescence of populations of Synechococcus and internal standard beads were obtained from the two-parameter histograms. When examining cells with extremely low PE contents, the utility of the blue/green excitation ratio measurement is limited by the sensitivity of the flow cytometer to fluorescence excited by light at 5 15 nm (Olson et al. 1988), which is about midway between the PUB and PEB absorption maxima. Populations with cells whose green-excited fluorescence was undetectable were therefore not included in the analysis of blue/green excitation ratios. Populations for which the blue/green excitation ratio data were not reliable constituted 23% (138/6 10) of all those sampled, and almost all were in surface waters in the oligotrophic North Atlantic. Studies with Synechococcus cultures- Cultures used for laboratory studies were obtained from John Waterbury or were isolated during the cruises. Cultures were grown in continuous light ( cool white fluorescent) at 22 C in f/2 medium (Guillard 1975) unless otherwise indicated. Results and discussion Distribution and abundance of Synechococcus- Synechococcus was present at every station we studied. As has been documented by others (Waterbury et al. 1979; Glover 1985), cell concentrations were highest near the coasts and lowest in the central oligotrophic ocean (Fig. 1). Surface concentra-

4 48 Olson et al I --- -J log surfacecellsml -l Fig. 2. Concentrations of Synechococcus integrated over deoth as a function of cell concentrations at the surface for each station at which depth profiles were analyzedl. The regression equation is y = ~; r2 = 0.68, n = 81, P < tions and integrated water-column concentrations were significantly correlated (Fig. 2) reflecting in part a lack of pronounced structure in most of the depth profiles (see below). The integrated cell abundance at each station was inversely related to the depth of the nitrite maximum layer (Fig. 3), which is a measure of the depth of the euphotic zone and an indicator of the relative oligotrop hy of the water column (Voituriez and Herbland 1977; Herbland and Voituriez 1979). The depth range over which Synechococcus was abundant was related to the depth of the nitrite maximum (Eq. 1) and the chlorophylll maximum (Eq. 2), but was not significantly correlated with the depth of the surface isothermal layer (Eq. 3): Z = (Z,); r2 = T60, n = 53 (P < 0.005) (1) = (2,); $?;:j6 y2 = 61 (P < 0.005) (2) Z = (2,); y2 = 531, n = 47 (P > 0.05) (3) where Zsyn is the depth at which cell concentration declined to 1 le of the surface value at a given station, Z, the depth of the nitrite maximum, which reflects the euphotic zone depth, ZC the depth of the chlorophyll maximum, and Z, the depth marking the bottom of the surface isothermal d Depth of Nitrite Max (ml Fig. 3. Synechococcus cell concentrations in the surface waters (0) and numbers integrated over the euphotic zone (0) as functions of the depth ofthe nitrite maximum for each station where nitrite was measured. The depth of the nitrite maximum is used as a measure of the euphotic zone. The regression equations for the surface and integrated cases are y = lx; y2 = 0.64, n = 57, P < and y = ~; r2 = 0.49, n = 57, P -c layer. Distinct subsurlace maxima in cell concentrations were observed in only 36 of 81 profiles examined; they occurred between 0 and 90 m, and both uniform cell distributions and subsurface maxima occurred at both open-ocean and coastal stations. Depths of those subsurface maxima present were not related to Z, as found by Murphy and Haugen (1985), nor were they related to ZN. As observed by Li and Wood ( 1988) subsurface Synechococcus maxima were consistently more shallow than the Z,-, but the two were not Cgnificantly correlated. If we consider the entire data set, the most consistent feature in the cell concentrationdepth profiles is that at every station there were at least 2.5 x 1 O,S cells ml- at depths about midway between the surface and the nitrite maximum; above these depths some stations had fewer than 2.5 x lo3 cells, while others had more (Fig. 4). It seems that the question of subsurface maxima may alternatively be stated as why do some stations have near-surface minima in cell concentrations? The answer could lie in different grazing pressures among sites or in the nutrient supply (i.e. mixing) history of the sites. Although we did find that the depth range over which Synechococcus was abundant

5 Synechococcus characteristics 49 (Z,,) was related to the depth of the nitrite maximum (Z,) (Eq. 1), the maximal depth at which cells were found in a given profile (i.e. the bottom of their range in the euphotic zone) bore no apparent relation to nitrite. In the clearest open-ocean waters (where Z, was m), there were very few Synechococcus cells present at the depth of the nitrite maximum, although shoreward (where Z, was as shallow as 40 m), the range extended to well below it (Fig. 4). This pattern probably reflects differing wavelength dependencies between the mechanisms responsible for the formation of the nitrite maximum layer, such as photoinhibition of nitrifying bacteria by near- UV and blue light (Olson 1981) and the absorption of light by Synechococcus (in the blue-green and green). The nitrite maximum might thus be expected to be deeper, relative to Synechococcus activity, in blue oceanic waters than in greener coastal waters. Distribution of phycoerythrin chromophore types- We have shown previously (Olson et al. 1988) that all the Synechococcus populations sampled during the transect across the North Atlantic contained phycoerythrin with both phycourobilin (PUB) and phycoerythrobilin (PEB) chromophores. The former have an absorption peak at about 495 nm (Ong et al. 1984) whereas the latter absorb primarily at 545 nm (Waterbury et al. 1986). Measurements of the blue/green excitation ratio of phycoerythrin fluorescence showed that most populations had relative PUB contents similar to or higher than that of any cultured isolate. Campbell and Iturriaga (1988) came to a similar conclusion for the Sargasso Sea on the basis of single-cell microscopy. At some coastal stations we found two or more populations of Synechococcus whose PE fluorescence intensities (excited by 488 nm light) were distinctly different; spectrofluorometric analysis of these bright and dim cells and their blue/green excitation ratios (from flow cytometry) showed them to have high and low PUB contents, respectively (Olson et al. 1988). We emphasize that total PE in the bright and dim cells may be equal. The difference in fluorescence intensity is due to differences in spectral absorption between the two types such that 0 Coastal Q--- sm 34 --o- Sm a--- Stn 38 w Sm 39 --*:--- Sm 43 Open Ocean B 2 I - I I - I log Cells ml-r Fig. 4. Concentrations of Synechococcus as a function of depth relative to that of the nitrite maximum for the two groups of stations indicated in Fig. 1. Profiles were taken each dawn (dashed lines) and dusk (solid lines) and were about 120 km apart. A. The first seven stations off the U.S. coast. B. Five consecutive open-ocean stations in the eastern North Atlantic. Symbols denote (reading down) stations from west to east. In coastal waters, the nitrite maximum could be a shallow as 40 m, whereas in the open ocean it was generally at 140 m or deeper. The upper isothermal layer included the first two or three depths in each profile. the bright population from 488-nm excitation can bc the dim population when excited at 515 nm. Analyses of samples from subsequent cruises in the Gulf of Mexico, the Caribbean, the Sargasso Sea, and the Pacific revealed that many, though not all, of the near-coastal stations had bright and dim populations co-occurring (cf. Fig. 5 with Fig. 1). In all cases in which we analyzed them

6 Olson et al. Fig. 5. The locations of stations where two distinct Synechococcus populations dliffering in PE fluorescence emission intensity (488-nm excitation, nm emission) were detected. Note that dual populations are found Ipredominantly in coastal waters (cf. Fig. 1). with dual-beam flow cytometry, the blue/ larger and less abundant than Synechococgreen ratios of the bright and dim cells were cus), also has a PUB/I?EB ratio about twice likewise high and low (Fig. 6), indicating the that of WH8 103 (J. Waterbury pers. comm.); presence of high- and low-pub cells. Ob- our extremely high PUB contents are thus served values of the blue/green excitation ratio for all populations studied to date span a large range (Fig. 6), but two main clusters Relative PE Fluorescence of pigment types can be seen clearly: low- (488 SC4515 ax!) Ratio PUB types similar in PUB content to the 1 10 a 1 L -- - cultured strain WH7803, and high-pub 0 types similar to strain WH the culturedi strain having the highest relative PUB content. In addition we note that low-pub cells are restricted to shallower depths than 50 high-pub cells. Low-PUB cells are rarely 3 found except in conjunction with high-pub cells: of 8 1 stations sampled, 34 had both 5 g loo high- and low-pub populations, and of the 47 stations with only a single population type,, 41 were of the high-pub type. Only WH7BO3 WI&o3 Low 'PUB High F'UB six stations had low-pub cells in the ab- -_- \L \L sence of high-pub ones. 200 I The highest blue/green excitation ratios Fig. 6. The blue/green excitation ratio (488 exe/ 5 15 exe) for phycoerythrin (PE) fluorescence of we observed corresponded to PUB contents Synechococcus populations as a function of depth. Valabout twice as high as that of WH ues for laboratory strains with either high or low phycothe high-pub strain of Synechococcus- and urobilin (PUB) are shown for reference. Mean values all occurred in the oligotrophic North At- of the dim populations at stations at which more than one cell type was detected based on 488-nm excitalantic. It is interesting to note that Synetion-0; means of the corresponding bright populachocystis, a planktonic blue-green found in tions-cl; stations with single population types-o. tropical oceans (but which is considerably

7 Synechococcus characteristics 51 I I I llllll I I I I 11 Relative FLS Fig. 7. A. Two populations of Qnechococcus with differing PE fluorescence intensities are clearly visible (December 1985 from a surface sample, N, W). B. Three populations are visible with some degree of overlap (September 1986 from 20-m depth, 39 I S N, w). C. Multiple populations appear to be present, but overlap significantly in fluorescence intensity (September 1986 from 32-m depth, N, 67O13.4 W). still within the documented range for phycocrythrins in nature. Although the general patterns described above were reasonably universal, in a few coastal samples we saw three distinct populations co-occurring and also populations whose fluorescence distributions were wider than usual, suggesting the presence of overlapping populations (Fig. 7). This kind of complexity is perhaps to be expected considering the variety of isolates presently in culture, and it almost certainly contributes to the spread in blue/green excitation ratios we observed, since we considered here only the means of such population distributions. To better understand the variability shown in Fig. 6, we analyzed a series of five profiles collected over 6 d at a single location near Santa Catalina, off southern California (Fig. 8). Like the overall data set, the data reveal no systematic trends in the blue/green excitation ratio with depth. The ratios varied twofold at any given depth over 6 d. This variability is not due to measurement error because replicate analyses from a given water sample gave results identical to within a few percent. The variation between profiles must result from changing water masses containing genetically different populations with different PUB contents, or physiological changes in the existing populations over time, resulting in changes in the blue/green excitation ratio. E B CI Relative PE Fluorescence Ratio (488 exe /515 excl 10 1 I..._.<.. wh7803 WI-i8103 *. tow PUB Hi& PUB v v Fig. 8. Blue/green excitation ratio for Synechococcus as a function of depth at a single station in the Southern California Bight ( N, 1 I W) sampled over 6 d (12-l 7 December 1986). The depth profiles are represented in chronological order as shown in key, Background dots are the data from Fig. 6 shown for comparison. Note that this station was one of the coastal stations at which only high-pub cells were present.

8 52 Olson et al. Table 1. Effects of nitrogen starvation on the relative fluorescence characteristics of six strains of Synechococcus. Cultures were grown in f/2 medium, split, and aliquots transferred into N-free medium, with flow cytometry of both kinds of cultures 2 d after cell division had ceased in the N-free cultures. Phycoerythrin fluorescence excited by 488-nm light- PE, the ratio of PE fluorescence excited by blue: (488 nm) and green (5 15 nm) light-b/g. Absorbance ratios (A495/A545) are from published values (Waterbury et al. 1986). -- N replete N starved N starved/n replete Stmin A495/A545 PE B/G PE B/G PE B/G oo ~---~ Although we have no firm evidence to support either of the hypotheses above, we can olffer some insights. We do know that Synechococcus clones with well-defined pigment compositions persist in culture with very little drift, so the genetic component is important (Waterbury et al. 1986). Variability of the PEB/PUB ratio in response to changing growth conditions within a given strain has not been systematically examined, and there is some conflicting evidence. We have shown previously (Olson et al. 1988) that the growth light intensity does not greatly influence the blue/green excitation ratio of WH8 103 and WH7803, the two cultured strains of Synechococcus we have used for reference in our fieldwork. At least two other strains have been shown, however, to vary their PUB/PEB ratios by up to twofold as a function of changing light conditions (Waterbury et al and pers. comm.). Moreover, we have recently examined the change in the pigment composition of six strains of Synechococcus as the cells become nitrogen starved in stationary phase (Table 1) and observed increases in the blue/green excitation ratio in five of the six N-starved cultures of up to 43%; it is interesting to note that the direction of change in excitation ratio we observed in N-starved cells was opposite to that in highlight-stressed cells (Olson et al. 1988), although both conditions caused decreased PE fluorescence. It is interesting that our observations as a whole did not reveal systematic changes in blue/green excitation ratio with depth. This lack of pattern suggests that field populations of Synechococcus were not accli- mating or adapting to depth in terms of spectral absorption properties, even though the same populations exhibited dramatic changes in absolute pigment levels with depths (see below). We should note, however, that the limits of-detection of the flow cytometer for 515-nm excitation (see methoals) make the analysis of cells from extremely nutrient-impoverished surface waters difficult; spectral acclimation may be important in these waters. Distribution of mean phycoerythrin jkorescence intensity- Phycoerythrin Auorescence intensity (when excited at 488 nm) of near-surface cells was typically much higher in coastal waters than in oligotrophic regions, whereas the fluorescence intensity of the cells in the deeper waters was generally the same, regardless ol the sampling site (Fig. 9A, B). The enhanced fluorescence in near- surface coastal populations probably reflects, in part, the greater availability of nutrients in these regions relative to the oligotrophic open ocean (Table 1). An additional factor to be considered here, however, is our choice of 488-nm excitation for our routine analyses. This wavelength is more efficiently absorbed by PUB than PEB; thus high-pub cells fluoresce more than low- PUB cells for a given amount of phycoerythrin. The brightness of cells in the upper waters, when excited1 by 488-nm light, was indeed related to their relative PUB content (i.e. their blue/green excitation ratio) for those populations whose excitation ratios we could measure reliably (Fig. 9C). It is important to note that this trend does not extend to the dimmest cells in our data set, which were found in the surface waters

9 Synechococcus characteristics Relative PE Fluorescence (488 exe) of the oligotrophic open ocean. Although we are unable to measure the green excitation in these cells (see methods), we know that they are not low-pub type cells. Therefore we conclude that fluorescence intensity in these cells is determined almost exclusively by PE content, which is low because of severe nutrient limitation. In summary, phycoerythrin fluorescence intensity depends on pigment type as well as the absolute amount of pigment, and the effects of pigment type are most obvious in welllit waters with adequate nutrient supply. As was observed by Li and Wood (1988), cell PE fluorescence increased at depth at both coastal and open-ocean stations, reflecting acclimation to lower light levels (Fig. 9). Because of their reduced fluorescence at the surface, this increase was much more dramatic in open-ocean populations, in some cases increasing by > 1 OO-fold. Note that the maximal PE fluorescence per cefi was about the same at the coastal and oceanic stations, and the depth of maximal fluorescence per cell was closely related to the depth of the nitrite maximum in both groups (Fig. 9). In the open oceans, cells were not usually found deeper than this level, whereas at coastal stations they were; the PE fluorescence of cells deeper than the nitrite maximum often decreased again, suggesting that they were not healthy. At many stations, the fluorescence per cell was constant throughout the mixed layer (Fig. 9A), indicating that the mixing rate was greater than the rate at which the cells could change their pigment content in acclimating to the changing light regime. At some stations, however (particularly in the open ocean), fluorescence per cell increased systematically with depth even within the mixed layer (Fig. 9B). These contrasting profiles suggest that PE fluorescence could Relative PE Fluorescence Ratio (488 e&515 exe) Fig. 9. A, B. Mean PE fluorescence of Synechococcus at the coastal and open-ocean stations described in Fig. 4. C. PE fluorescence as a function of B/G excitation ratio for populations in the near-surface layer from the entire data set. (Data from deeper waters were excluded to avoid the confounding effects of photoacclimation.) The near-surface layer was defined as the shallower of half the depth of the nitrite maximum or the depth to which fluorescence remained constant to within a factor of 2 from one sample depth lo the next. The large squares represent data from the coastal stations used as examples in panel A. The regression equation was y = x; r2 = 0.50, y1 = 130, P <

10 54 Olson et al shiftup i lo PE 0 -I-I- - * Time After Shift (h) Fig. IO. Experiment designed to estimate photoacclimation time of Synechococcus. Exponential cultures of Synechococcus strain WH7803, growing in continuous illumination at either 315 or 33 PEinst m-2 s-l, were each shifted to the opposite light regime at time = 0. PE fluorescence and FLS of individual cells were then followed with flow cytometry as the cultures adapted to the new conditions. A. When light intensity was lowered from 315 to 33 PEinst m-* s-l, the mean PE fluorescence of the population began to increase immediately, but mean FLS showed little change. After 50 h, mean PE iluorescence had not yet reached llthe acclimated level for cells grown at 33 PEinst m-2 s-l. B. When light intensity was raised from 33 to 315 PEinst m-* s-l, FLS increased and PE fluorescence declined. FLS eventually returned to the original level after 30 h when the cells had fully acclimated to the new light level. mation in Synechococcus (Fig. 10) and have found that cells can fully adapt to a lo-fold increase in light intensity over a period of 30 h but require >50 h to acclimate to a decrease in light intensity of the same magnitude. We can use these results to make some crude inferences about mixing rates in the surface layer of the open-ocean stations, in.which PE fluorescence per cell did not appear to be homogenized by mixing (Fig. 9B). If we examine the data from the top 40 m (the uppermost two or three points) of each profile (representing roughly an order of magnitude range in light intensity, similar to the lab experiment), we see that PE fluorescence per cell at the bottom of the layer was about 1.6 times higher than that at the surface. In a stable water column, we would expect to find a fourfold increase in fluorescence, based on the lab experiment. This disparity reflects the difference between the time scales of mixing and photoadaptation and suggests that PE fluorescence profiles may be useful in modeling the mixing of phytoplankton. Admittedly, these calculations assume (among other things) that the natural populations behave similarly to pure cultures of undoubtedly different strains, that the kinetics of photoacclimation are the same regardless of initial and final conditions, and that nutrient limitation does not affect acclimation. Still, they illustrate that the kine tics of photoacclimation are clearly of the right order of magnitude for most mixing events. In the future, shift-up and shift-down experiments will be done with natural populations under field conditions, providing more rigorous characterization of photoacclimation for input to a mixing model. The ubiquitous distribution of Synechococcus in surface waters of the ocean suggests that this system might prove widely applicable for measuring physical processes in the mixed layer. be used to calculate mixing rates in surface Distribution of mean cell size forward light waters in the same way as proposed for oth- scatter) -Light scattering by particles is not er photoadaptive processes (Harris 1980; simple to interpret; its angular intensity dis- Lewis et al. 1984; Welschmeyer and Hoepff- tribution at a given wavelength is deter- ner 1986; Cullen and Lewis 1988). We have mined by a complex interaction between done simple laboratory experiments de- size, shape, and refractive index (Van de signed to put a time scale on photoaccli- Hulst 1957; Ackleson and Spinrad 1988).

11 Synechococcus characteristics 55 O-?I : Dawn Relative FLS 1. s b2 l- 5i & stn 34 B 8 --t-t&n Stn m-stn dk-- SfJl 43 Open ocean B Relative FLS Fig. 11. A, B. Mean forward light scatter of Synechococcus at the coastal and open-ocean stations described in Fig. 4. C. Detail of the upper part of the profiles shown in panel B. Nonetheless, empirical tests suggest that FLS is systematically related to cell size for many kinds of eucaryotic phytoplankton (Olson et al. 1989), and FLS of Synechococcus cells is at least roughly proportional to cell volume as measured with a Coulter counter (data not shown). Mean FLS for surface Synechococcus populations showed a fourfold range in our entire data set, with the greatest variation found in coastal waters. The populations with highest and lowest FLS were both found at coastal stations. Overall, there was no significant relationship between mean FLS and water type. FLS of Synechococcus populations also varied with depth, with the deepest cells in nearly all cases being much larger than those in the upper waters (Fig. 11). On several occasions, WC filter size-fractionated samples from different depths to determine whether the FLS signal was indeed reflecting cell size. In these cxperiments we observed consistently an increasing proportion of the Synechococcus retained by a l-pm Nuclepore filter as depth increased (data not shown), as has also been observed by Li and Wood (1988). These observations are consistent with microscopic examination (Waterbury et al. 1986; Glover 1985). The increase in FLS with depth typically began at the same depth as the decline in cell concentration and the increase in PE fluorescence per cell. It is noteworthy that FLS did not increase at depth as dramatically as did PE fluorescence; the maximal increase in FLS was only about sixfold, as opposed to more than loo-fold for PE fluorescence. Thus cells at depth are not only larger than cells at the surface, but also have higher PE fluorescence per unit of cell volume. For reasons that are unclear, laboratory cultures do not appear to exhibit these dramatic increases in cell size under low light (Fig. 1 OA). Even in cultures acclimated to a light gradient spanning two orders of magnitude, only a 40% increase in cell volume is observed at the low end (Kana and Glibert 1987). We observed surprisingly clear diel patterns in FLS in cells from surface populations sampled from vast regions of the ocean over many days (Fig. 12A, B). A single station in the Sargasso Sea sampled repeatedly over 24 h yielded similar but much smoother data, as we might expect (Fig. 12C). The changes in cell size, which superficially re-

12 V.&V A 0 Puerto Rico to WCKXIS Hole * * B North Atlantic Olson et al Local Time fh) Fig. 12. Mean forward light scatter of Synechococcus cells from surface waters as a function of the time of day during a transect from Puerto Rico to Woods Hole, Massachusetts (December 1985) a transect across the North Atlantic (September 1986), and a 24-h station in the Sargasso Sea (September 1987). The data were fitted to a third-order polynomial by least-squares. Note that all three data sets exhibit maxima at about 1800 hours and minima at about 0600 hours local time. semble patterns in mitotic index reported for Synechococcus in the Sargasso Sea (Waterbury et al. 1986), apparently reflect increasing size of the cells as they photosynthesize and grow during daylight. The diel patterns in FLS were not limited to surface waters, but #extended well below the mixed layer at many stations (see Fig. I 163, C, in which only the uppermost two or three points of each profile were in the mixed layer). Closer examination of depth profiles of mean FLS suggests that cells were sometimes larger at the surface than at middepth, especially at dusk. Using the Atlantic transect, for which we had 12 d of dawn and dusk depth profiles, we compared the FLS of cells half way between the surface and the nitrite maximum (FLS,) with those of cells at the surface (FLS,): On 10 of the 12 d the ratio FLS, : FLS,, was greater at dusk than at dawn. If, as we suspect, the FLS increases we observe reflect cell growth, this pattern suggests that cells near the surface are growing faster than those at middepth. Not only does the diel change in FLS appear to reflect changes in growth rate with depth, but it also appears to reflect variations in growth rate between the open ocean and coastal populations. It was about 20-50% among the more! open-ocean stations (Fig. 11 B, 12) and about 80% among coastal stations (Fig. 11 A). In order to translate these changes to a specific growth rate for the populations we would have to know something about the degree of synchrony in cell division. Laboratory studies of Synechococcus populations grown on diel photocycles (Waterbury et al. 1986; Chisholm et al. 1986; Armbrust et al, 1989)~ indicate that they do not synchronize to the photocycle. Rather, the populations grow exponentially during the light period and arrest in their cell cycles during the dark. This growth mode makes the calibration of our diel patterns in light scatter difficult without some independent measure of growth rate. It may be useful, however, for looking at comparative growth rates of populations and shows promise for future applications. In addition, the similarity between the pat terns we observed and diurnal variations in beam attenuation coefficient such as those reported by Siegel et al. (1989) emphasizes the importance of

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